RNA polymerase II transcripts (heterogeneous nuclear RNA) are associated with nuclear ribonucleoprotein particles (hnRNP). These particles may have a central role in mRNA processing. In this application, we seek continued support for a program on hnRNP structure and function. The proposed experiments make use exclusively of specific gene transcripts, rather than total hnRNP. These studies involve in vitro transcription of cloned genes by RNA polymerase II, combined with our recent successful in vitro assembly of specific hnRNP particles. The research also exploits new techniques we have developed for probing RNA-protein and RNA-RNA interactions. Three main lines of investigation will be pursued regarding hnRNP function: (1) Do hnRNP proteins have specific DNA-binding properties?, (2) does the nascent transcript interact with the template and do hnRNP proteins attenuate this process?, and (3) is hnRNP structure important for splicing? hnRNP structure will be studied with emphasis on sequence-specific aspects, as well as vicinal interactions of small nuclear RNA-associated proteins with the hnRNA strand.